ORF22-04 - Selective Overexpression of Makorin Ring Finger Protein 3 in Kiss1 Neurons Delays Pubertal Onset in Male and Female Mice

Background: Makorin ring finger protein 3 (MKRN3), an upstream inhibitor of GnRH release, is an important regulator of the age of menarche; loss-of-function mutations in MKRN3 are the most common genetic etiology of central precocious puberty. In mice, expression of Mkrn3 in the hypothalamic arcuate nucleus (ARC), where kisspeptin, neurokinin B and dynorphin (KNDy) co-expressing neurons are located, is high in early life and declines before pubertal onset. KNDy neurons are critical for pubertal onset and maintenance of reproduction. Neonatal hypothalamic overexpression of Mkrn3 leads to delayed puberty in female mice and decreased neurokinin B and kisspeptin protein levels in the ARC peripubertally. However, it is unclear if the effects on these neuronal populations are direct and/or indirect. Therefore, we hypothesized that Mkrn3 inhibits Kiss1 neurons directly; thus, selective overexpression of Mkrn3 in Kiss1 neurons would result in suppression of the HPG axis, manifested as delayed puberty in mice.

Methods and

Results: We generated a novel transgenic ROSA26-Mkrn3 mouse, which has selective, targeted Mkrn3 overexpression in Kiss1-expressing cells when bred with Kiss1-Cre mice [Tg(Kiss1-cre)J2-4Cfe]. The ROSA26-Mkrn3 mouse model contains a strong constitutive promoter, CAG, and a STOP sequence (loxP-neomycin-polyadenylation-loxP), which is removed by homologous recombination in the presence of Cre-recombinase. Kiss1-Cre+/-Rosafl/fl male mice were bred with ROSA-Mkrn3fl/fl female mice to generate the experimental group: Kiss1Cre+/-Rosafl/fl (Kiss1Mkrn3-OE); and the control group: Kiss1-Cre-/-Rosafl/fl (Kiss1Control). Initial validation of the model was performed by RNA-FISH, which demonstrates colocalization of Mkrn3 and Kiss1 mRNA in AVPV neurons in Kiss1Mkrn3-OE but not in Kiss1Control gonad-intact adult female mice. Similar to our prior mouse model of neonatal hypothalamic overexpression of Mkrn3, body weight was unaffected in both sexes from postnatal day (PND) 21 to 91. Kiss1Mkrn3-OE male mice (n=21) had delayed preputial separation compared to Kiss1Control mice (n=12; p= 0.01). In Kiss1Mkrn3-OE female mice, age at vaginal opening did not differ (p=0.22; n=19); however, first estrus was delayed compared to Kiss1Control mice (p < 0.01; n=12). Subsequent estrous cyclicity was normal from PND 60 to 90. Body weight at time of pubertal onset did not differ in males or females.

Conclusions: The delayed puberty phenotype in the setting of Mkrn3 overexpression in Kiss1 neurons provides further support for the important action of Mkrn3 on KNDy neurons. These findings contribute additional evidence that Mkrn3 can exert inhibitory effects on the reproductive axis in mice beyond the juvenile period, which has implications for Mkrn3 as a potential therapeutic target.